Rac-deficient cerebellar granule neurons die before they migrate to the internal granule layer

Granule neurons are the most common cell type in the cerebellum. They are generated in the external granule layer and migrate inwardly, forming the internal granule layer. Small Rho GTPases play various roles during development of the nervous system and may be involved in generation, differentiation and migration of granule neurons. We deleted Rac1, a member of small Rho GTPases, by GFAP-Cre driver in cerebellar granule neurons and Bergmann glial cells. Rac1flox/flox; Cre mice showed impaired migration and slight reduction in the number of granule neurons in the internal granule layer. Deletion of both Rac1 and Rac3 resulted in almost complete absence of granule neurons. Rac-deficient granule neurons differentiated into p27 and NeuN-expressing post mitotic neurons, but died before migration to the internal granule layer. Loss of Rac3 has little effect on granule neuron development. Rac1flox/flox; Rac3+/−; Cre mice showed intermediate phenotype between Rac1flox/flox; Cre and Rac1flox/flox; Rac3−/−; Cre mice in both survival and migration of granule neurons. Rac3 itself seems to be unimportant in the development of the cerebellum, but has some roles in Rac1-deleted granule neurons. Conversely, overall morphology of Rac1+/flox; Rac3−/−; Cre cerebella was normal. One allele of Rac1 is therefore thought to be sufficient to promote development of cerebellar granule neurons.


5-Bromo-2′-deoxyuridine (BrdU) labeling.
To assess the cell-cycle withdrawal, differentiation and migration of cerebellar granule neurons, mice were injected with 25 mg/kg of BrdU intraperitonially on postnatal day 7 (P7) and sacrificed 24, 48 and 72 h after the injection. Cerebella were fixed in paraformaldehyde and embedded in paraffin. Sagittal sections were subjected to immunohistochemistry mentioned above.
Statistical analysis. For quantitation of the immunohistochemical results, 3 animals (2 sections/animal) were examined in each genotype. Immuno-positive signals were analyzed using ImageJ (https:// imagej. nih. gov/ ij/). Statistical analyses were carried out by one-way ANOVA followed by a Tukey's post hoc test. Jarque-Bera test was used to determine whether sample data were normally distributed. Differences were defined as statistically significant when P < 0.05.

Results
Deletion of Rac in cerebellar granule neurons results in loss of granule neurons. In this study, Rac1 was deleted using GFAP-Cre mice, which induces Cre/loxP recombination in cerebellar granule neuron precursors from embryonic day 14.5 and also in Bergmann glial cells ( Supplementary Fig. 1) [13][14][15][16] . At birth, cerebella of Rac1 flox/flox ; Cre mice were slightly smaller than those of Rac1 +/flox ; Cre mice ( Fig. 1A). The difference in cerebellum size became larger as the mice grew up, and cerebella of Rac1 flox/flox ; Cre mice were markedly smaller than those of Rac1 +/flox ; Cre mice on P14. Rac3, a close homolog of Rac1, is expressed in the nervous system 4 . Although Rac3-knockout mice showed no obvious histological abnormalities in the brain 5 , Rac3 may play some roles in the Rac1-deleted brain, Rac1/Rac3 compound knockout (Rac1 flox/flox ; Rac3 −/− ; Cre) mice were therefore generated. The cerebella of Rac1 flox/flox ; Rac3 −/− ; Cre mice were much smaller than those of Rac1 flox/flox ; Cre mice on P14 (Fig. 1A). Sagittal and coronal sections revealed that not only anterior medial parts, but also caudal lateral parts of the cerebella were affected in Rac1 flox/flox ; Rac3 −/− ; Cre mice. Immunohistochemistry showed NeuNpositive cerebellar granule neurons were almost completely absent in Rac1 flox/flox ; Rac3 −/− ; Cre mice (Fig. 1B). The phenotype of Rac1 flox/flox ; Rac3 +/− ; Cre mice was milder than that of Rac1 flox/flox ; Rac3 −/− ; Cre mice ( Fig. 1A,  B). Presence of at least one allele of Rac1 appeared sufficient to maintain cerebellum structures because overall morphology of the cerebellum of Rac1 +/flox ; Rac3 −/− ; Cre mice was normal (Fig. 1A, B). Rac is suggested by these results to be essential for the development of granule neurons in the entire cerebellum. Loss of Rac3 itself has little effect on development of cerebellar granule neurons, but it can play some roles in Rac1-null situation.     (Fig. 3C, arrowheads). NeuN-positive cells that cannot migrate into the molecular layer may therefore differentiate into cells similar to granule neurons in the IGL in the EGL.
Rac-deficient granule neurons die before they migrate to the IGL. To confirm whether Rac-deficient granule neurons differentiate as far as NeuN expression and die in the inner most part of the EGL, mice were injected with BrdU on P7 and differentiation and migration of BrdU-incorporated granule neurons were examined. A large volume of the BrdU-incorporated granule neurons differentiated to NeuN-positive cells in 48 h in all the genotypes (Fig. 4A). However, significantly fewer BrdU-incorporated cells migrated to the IGL (past the Purkinje cell layer) in Rac1 flox/flox ; Cre, Rac1 flox/flox ; Rac3 +/− ; Cre and Rac1 flox/flox ; Rac3 −/− ; Cre mice compared with other genotypes at 48 h (Fig. 4A) and 72 h (Fig. 4B). Only a few BrdU-incorporated cells survived in Rac1 flox/flox ; Rac3 −/− ; Cre mice at 72 h (Fig. 4B). In addition, some BrdU-positive cells in the EGL of Rac1 flox/flox ; Cre, Rac1 flox/flox ; Rac3 +/− ; Cre and Rac1 flox/flox ; Rac3 −/− ; Cre mice were also positive for cleaved Caspase-3 (CC3), an apoptotic cell marker, at 48 h after the BrdU injection (Fig. 4C, arrowheads). The proportion of CC3 + /BrdU + cells among all BrdU + cells was significantly increased in these mice. Rac-deficient granule neurons can therefore differentiate almost normally as far as NeuN expression but they die before they migrate to the IGL. Migration of granule neurons was impaired in Rac1 flox/flox ; Cre and Rac1 flox/flox ; Rac3 +/− ; Cre mice, and the phenotype was much more severe in Rac1 flox/flox ; Rac3 +/− ; Cre mice than that in Rac1 flox/flox ; Cre mice, suggesting that Rac is required for the migration of granule neurons and that Rac3 has some roles in Rac1-deficient granule neurons in terms of migration. Existence of NeuN-positive cells between the EGL and IGL in Rac1 flox/flox ; Cre and Rac1 flox/flox ; Rac3 +/− ; Cre mice also suggests impaired migration of granule neurons in these mice (Fig. 4A, B).

Loss of Rac caused disorganization of processes of Bergmann Glia.
The GFAP-Cre mouse line used in this study induces Cre/loxP recombination not only in granule neurons, but also in Bergmann glial cells 15,16 . Several findings suggest that Bergmann glia works as a scaffold for migrating granule neurons 18 . The arrangement of processes of Bergmann glia was therefore examined by immunostaining for GFAP. GFAP-positive processes of Bergmann glial cells in Rac1 flox/flox ; Cre, Rac1 flox/flox ; Rac3 +/− ; Cre and Rac1 flox/flox ; Rac3 −/− ; Cre mice appeared disorganized probably due to hypoplasia/aplasia of the IGL (Fig. 5). We also immunostained Sox2, which labels nuclei of Bergmann glial cells 19,20  Rac-deficient granule neurons that cannot migrate into the molecular layer can differentiate into mature cells in terms of marker expression. However, it is difficult to determine whether granule neurons die because they cannot make complete differentiation or because they cannot migrate to the IGL and are mis-localized. The precise cause of the cell death of Rac-deficient granule neurons remains unknown. Small Rho GTPases including Rac are known to have important roles in the development of cerebellar granule neurons both in cell autonomously and non-cell autonomously. For example, deletion of RhoA or Cdc42 in granule neurons delayed their migration 17,22 . In Bergman glia, deletion of Cdc42, but not Rac1, impaired migration of granule neurons, but compound deletion of both Cdc42 and Rac1 much more severely impaired the migration of granule neurons than that of Cdc42-deletion alone 21 . Furthermore, in granule neurons, deletion of β-chimaerin, a GTPase activating protein (GAP) of Rac, or Trio, a guanine nucleotide exchanging factor (GEF) of RhoA, RhoG and Rac, caused defective migration 23,24 . Disruption of Abr and Bcr, RacGAPs, caused misalignment of Bergmann processes and granule cell migration defects 25 . These findings together with ours suggest that small Rho GTPases not only in granule neurons themselves but also in Bergman glial cells play very important roles in the development of cerebellar granule neurons, especially in their migration. The activity of each small Rho GTPase must be tightly regulated.  www.nature.com/scientificreports/ Deletion of both Rac1 and Rac3 in the cerebellum was also previously shown to cause severely impaired development of granule neurons 8 . The biggest difference between current and previous studies is the distribution of hypoplasia/aplasia in the cerebellum. In Atoh1-Cre; Rac1 flox/flox ; Rac3 −/− mice, agenesis of the IGL was observed only in the anterior medial part of the cerebellum 8 , but in our Rac1 flox/flox ; Rac3 −/− ; GFAP-Cre mice, it was observed in the entire cerebellum. Furthermore, degeneration of cerebellar granule neurons was much more severe in Rac1 flox/flox ; Rac3 −/− ; GFAP-Cre mice than in Atoh1-Cre; Rac1 flox/flox ; Rac3 −/− mice. Deletion of receptor for activated C kinase (Rack1) by GFAP-Cre driver resulted in much more severe phenotype than that by Atoh1-Cre driver 16 . Rack1 deletion by GFAP-Cre mice resulted in agenesis of lobules throughout the rostral to caudal parts of the medial vermis. On the other hand, loss of Rack1 by Atoh1-Cre mice affected only the rostral part of the cerebellum. This difference may be caused by the difference in Cre recombinase activity in GFAP-Cre and Atoh1-Cre mice. Cre recombinase activity in the cerebellum of Atoh1-Cre mice is higher in the rostral part compared with in the caudal regions 16 . By deletion of Rac1 in the entire cerebellum by GFAP-Cre driver, Rac was shown in our study to play crucial roles in the development of cerebellar granule neurons not only in the anterior medial part but in the entire cerebellum. Nakamura et al. 8 identified Mid1 as a downstream target of Rac to promote cerebellar development. The human ortholog is a responsible gene for Opitz G/BBB syndrome characterized by malformation of midline structures 26 . In addition, Mid1-deficient mice showed hypoplasia of the cerebellum in the anterior medal portion only 27 . Other downstream targets of Rac may play an important role in the caudal lateral part of the cerebellum.
Deletion of Rac1 in telencephalic neuroepithelium resulted in accelerated cell cycle exit of progenitors 28 . Conversely, deletion of Rac1 or Rac1/Rac3 in the medial ganglionic eminence reduced cell cycle exit of interneuron progenitors 29,30 . Nakamura et al. 8 and our group both showed no effect of deletion of both Rac1 and Rac3 in the cerebellum on cell cycle exit of granule neuron progenitors. Rac may play different roles in cell cycle exit of progenitors depending on cell types.
Rac plays a variety of roles during development of the nervous system, including neurogenesis, differentiation, migration, dendritogenesis, axon guidance and synapse formation 1,2 , but there is poor current understanding of downstream signaling pathways being involved in each process. In the central nervous system, Rac3, a close homolog of Rac1, is ubiquitously expressed, and it may have roles in Rac1-deficient neurons, making it difficult to recognize the functions of "Rac" and its downstream targets. The present study showed that deletion of both Rac1 and Rac3 in cerebellar granule neurons resulted in much more severe impairment in survival and migration than that of Rac1-deletion alone. Our experimental system can therefore be considered to be an effective tool for identification of downstream targets of Rac in neuronal survival and migration.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.